The related parent applications describe inventions which utilize the capacity of nucleic acids for self assembly to construct a series of unimolecular DNA foldbacks or HBO's that are good enzyme substrates. In these molecules a long block of DNA is linked through a tether to a complementary short block of DNA. The tether may consist of dT residues, biotin residues, dodecyl phosphate residues, aminopropyl phosphate residues, or trivalent residues which are similar to the above but will allow for side-chain modification. These modifications may include chemiluminescent, fluorescent, or biotin moieties. The tether promotes intramolecular hybridization of the two regions of complementary DNA to form foldbacks having a free 3' hydroxyl on the short DNA strand and an overhanging DNA strand at the end of a short DNA-DNA hybrid. Appropriate HBO's have been shown to be substrates for restriction enzymes, human DNA methyl transferase and DNA polymerase I from E. coli.
Relevant to this invention is the discovery that DNA polymerase I is effective in extending each of the tethered foldbacks to a discrete length corresponding to full extension of the short block in the foldback using the 5' overhang as a template. Variation in the type of tether used permits chromatographic discrimination between otherwise isomeric forms of the molecules.